Regulated exocytosis of secretory membrane vesicles proceeds via recruitment of the vesicles to the plasma membrane at specific sites conducive for exocytosis, followed by staged fusion into the plasma membrane. Generalized defects in regulated exocytosis cause single or multi-system disease in humans, and the promotion (e.g in diabetes) or inhibition (e.g. in anaphylaxis) of regulated exocytosis underlies numerous therapeutic modalities. Our general hypothesis is that manipulation of the iipid environment is a key element in this process. Published reports and our preliminary evidence suggest that the signal-transducing enzyme Phospholipase D1 (PLD1) plays a role late in this process during fusion of the vesicles into the plasma membrane via production of phosphatidic acid (PA), the Iipid product of PLD action. Many avenues of investigation are now timely to explore to determine the mechanism through which it functions. We propose to carry out the following specific aims to address these questions: 1. What are the temporal and spatial relationships of PLD1 activation and PA generation to their facilitation of regulated exocytosis? We will examine whether PLD1 activation is required acutely at the time of secretion, which of PLDVs activators participate in the PLD1-facilitated process, and where PA is produced during the fusion event. 2. How is regulated exocytosis facilitated by increasing levels of PA? We will use electron microscopy to examine the morphology of vesicles hindered from completing fusion in cells lacking PLD1 to determine which step the fusion process is blocked at. We will also examine potential mechanisms though which PLD1 and its product PA may be functioning, including regulation of the production of PI4,5P2 by stimulation of PI4P5KI, the enzyme family that generates PI4,5P2;recruitment to exocytic fusion sites of CAPS, a PI4,5P2-binding protein that is required for fusion;and potential affects on the fusion process itself though promotion of fusion pore formation or expansion. The results from the proposed experiments will substantially further our understanding of the mechanisms through which PLD1 promotes secretion during regulated exocytosis.